This proposed research will examine how adult taste stem cells generate mature taste cells, and will develop a culture system to grow, expand, and differentiate taste stem cells to study taste regeneration processes in vitro. The nature of taste stem cells is unknown although those in the posterior tongue express Lgr5; those in both the anterior and posterior express Lgr6.To expand our preliminary understanding of these cells, we propose to use genetic lineage tracing strategies and to establish a novel cell culture approach to further characterize Lgr5+ and Lgr6+ taste cells and determine their regenerative capacity both in vivo and in vitro. In this project, we will investigate two overlapping areas: In Aim 1, we will determine whether Lgr5+ and Lgr6+ cells are taste stem/progenitor cells in vivo. We will characterize the nature of Lgr5+ and Lgr6+ cells in detail using existing genetically engineered mice and various marker induction regimens. Subaims will determine whether (Aim 1.1) the strong Lgr5+ cells are taste stem cells for posterior tongue, (Aim 1.2) Lgr5+ cells give rise to Shh+ cells en route to mature taste bud cells, (Aim 1.3) individual taste buds are monoclonal (derived from a single progenitor cell) or oligoclonal (derived from a few progenitor cells) and Lgr5+ cells are multipotent (differentiate into different cell types) or unipotent (develop into ony one cell type), and (Aim 1.4) Lgr6+ cells represent adult taste stem/progenitor cells in anterior and posterior tongue. In Aim 2, we will determine the regenerative capacity of Lgr5+ and Lgr6+ cells in vitro. Single Lgr5+ intestinal stem cells are able to build crypt-villus structures in vito. We will isolate Lgr5+ and Lgr6+ stem cells from tongue by FACS sorting and culture them in defined medium in ultra-low attachment plates to examine their growth, expansion, and long-term passages (Aim 2.1), as well as the relatedness of Lgr5+ and Lgr6+ cells. Using a Matrigel-based 3-D culture system, we will determine the regenerative capacity of Lgr5+ and Lgr6+ cells, whether single Lgr5+ and Lgr6+ cells are multipotent or unipotent, and which culture components affect growth, expansion, and differentiation (Aim 2.2). These areas of study are significant first steps toward a regenerative approach for treating patients who suffer from taste impairment, such as cancer patients receiving head and neck radiation therapy or chemotherapy, and may lead to the development of a culture system for screening novel taste modulators.